2019-09-15T19:04:20Zhttps://ir.soken.ac.jp/?action=repository_oaipmhoai:ir.soken.ac.jp:000025142018-10-26T05:14:17Z00002:00430:00022Sleep/wakefulness regulation by controlling the activity of orexin neurons using optogeneticsSleep/wakefulness regulation by controlling the activity of orexin neurons using optogeneticsenghttp://id.nii.ac.jp/1013/00002423/Thesis or Dissertation常松, 友美ツネマツ , トモミTomomi, TSUNEMATSU総合研究大学院大学博士（理学）総研大甲第1444号2011-03-24Sleep, which is a conserved phenomenon not only in vertebrates but also in invertebrates such as drosophila, is one of the instinctive behaviors that occur in the brain. Sleep is thought to be a fundamental behavior for maintenance of life because sleep deprivation for a few weeks leads mice to death. Additionally we humans usually spend one third of our lives in sleeping. However, physiological function of sleep is less well understood so far. In the present study, to clarify the importance of orexin neuronal activity in the regulation of sleep/wakefulness, I introduced a novel technology ‘optogenetics’ using light-activated proteins and generated two different transgenic mice. I focused on especially the relationship with human sleep disorder narcolepsy (chapter I) and the neuronal circuit involved in sleep/wakefulness regulation (chapter II) using each transgenic mice.<br/><br/>Chapter I: Sleep/wakefulness regulation by controlling the activity of orexin neurons using orexin/Halo transgenic mice Orexin neurons have a crucial role in the regulation of sleep and wakefulness. Animals that lack the orexin neurons show phenotypes remarkably similar to that of the human sleep disorder, narcolepsy. Recent in vitro electrophysiological studies revealed the afferents to and efferents from orexin neurons. However, it is not completely understood how their activity induces wakefulness in vivo. To help determine how these neurons promote wakefulness, I generated transgenic mice in which orexin neurons expressed halorhodopsin, an orange light-activated chloride ion pump. Immunohistochemical analysis revealed that 94% of orexin-immunoreactive neurons specifically express halorhodopsin. Slice patch clamp recordings of orexin/halorhodopsin neurons demonstrated that photic illumination reduced orexin neuron discharge. Acute silencing of orexin neurons in vivo by illuminating orange light into the hypothalamus decreased electromyogram (EMG) power and increased slow wave components of electroencephalogram (EEG) power suggesting that the mice showed slow wave sleep (SWS). Photic illumination induced-SWS was time-of-day dependent, indicating that activation of orexin neurons is necessary for wakefulness during the light period. Although the discharge of dorsal raphe (DR) serotonergic neurons, which receive orexin neuronal excitatory input, was gradually reduced to almost the same level of SWS during acute orexin neuron photoinhibition, DR serotonergic neurons exhibited normal discharge rates in mice lacking orexin neurons during either sleep or wakefulness. These results indicate that serotonergic DR neuronal activity is usually highly dependent on orexin neuronal activity but can be regulated appropriately in the chronic absence of orexin input. This difference between acute vs. chronic reduction in orexin input may underlie the progression of symptomatology in narcoleptic patients.<br/><br/>Chapter II: Sleep/wakefulness regulation by controlling the activity of orexin neurons using orexin/Arch transgenic mice Orexin neurons have a crucial role in the regulation of sleep and wakefulness. Recent in vitro electrophysiological studies revealed the afferents to and efferents from orexin neurons. However, it is not completely understood how their activity induces wakefulness in vivo. To help determine how these neurons promote wakefulness, I generated transgenic mice in which orexin neurons expressed archaerhodopsin-3, a yellow-green light-activated proton pump. Immunohistochemical analysis revealed that about 80% of orexin-immunoreactive neurons specifically express archaerhodopsin-3. Slice patch clamp recordings of orexin/archaerhodopsin-3 neurons demonstrated that repetitive and long-lasting photic illumination could silence the activity of most of orexin neurons. Acute silencing of orexin neurons in vivo by illuminating yellow-green light for 1 min into the hypothalamus induced slow wave sleep (SWS) in the light period, but failed in the dark period. This result showed good agreement with the result obtained from Halo transgenic mice. To confirm that photic inhibition of orexin neurons failed to induce SWS in the dark period was not caused by insufficient inhibition of orexin neurons, I performed immunohistochemical study. c-Fos, a marker for activation of neurons, and orexin ware stained. Continuous photic inhibition of orexin neurons for 2 hr significantly decreased c-fos expression in the orexin neurons in the dark period compare with that without photic inhibition. Although photic illumination certainly silenced the activity of orexin neurons in vivo, photic inhibition of orexin neurons failed to induce SWS in the dark period. These results might suggest a necessity of sleep pressure to induce SWS. To study the effect of sleep pressure, mice were sleep deprived for 3 hr before the experiments. The mice received sleep deprivation for 3 hr showed SWS by photic inhibition of orexin neurons in the dark period. These results suggest that not only the cessation of the activity of orexin neurons but also the homeostatic sleep pressure is a critical factor to induce SWS in mice.<br/>https://ir.soken.ac.jp/?action=repository_action_common_download&item_id=2514&item_no=1&attribute_id=19&file_no=2CC BY-NC-ND2012-01-16